The evaluation of the Oxidative Stress (OS) condition assumes particular prognostic importance in the case of patients with cardiovascular diseases. When the excessive amount of biological molecules oxidised in the blood (serum or plasma) is not compensated by an adequate antioxidant capacity, OS is generated, which, if protracted over time, determines a reduction in life expectancy, particularly in patients with cardiovascular disease (Vassalle C. et al., “Elevated levels of oxidative stress as a prognostic predictor of major adverse cardiovascular events in patients with coronary artery disease”, J Atheroscler Thromb. 2012; 19(8):712-7). In the human and animal body, the measurement of hydroperoxides (ROOH) [where R represents a molecule having a chemical nature other than simple hydrogen up to macromolecules such as lipids, glycerophospholipids, proteins, carbohydrates, DNA, RNA], is considered among the most reliable markers of OS [Cornelli U. et al., “The Oxidative Stress Balance Measured in Humans with Different Markers, Following a Single Oral Antioxidants Supplementation or to Diet Poor of Antioxidants”, Journal of Cosmetics, Dermatological Sciences and Applications, 2011, 1, 64-70].
ROOH are the expression of almost all biological macromolecules at the initial stage of the oxidation process in that they represent the condition common to all molecules capable of propagating the OS once they come into contact with Fe2+ (ubiquitous metal) by means of the known ‘Fenton reaction’:Fe2++ROOH→Fe3++ROH+.OH
This reaction brings about the formation of a hydroxyl radical (.OH), which is by far the most powerful oxidant in the human body, being provided with a reaction half-life in the order of 10−9 seconds. Since ROOH are relatively stable, they circulate freely in both the intracellular and extracellular blood and interstitial context, with the characteristic of carrying their oxidative potential away from the point of formation. It is this very latter characteristic that makes them dangerous diffusers of OS that are far more harmful than the other, more rapidly reactive species (e.g. ROS or RNS, respectively oxygen- or nitrogen-reactive species) having local/immediate action (such as O2. or .OH, respectively superoxide and hydroxyl radical).
The hypothesis of the role of lipids and oxidised lipoproteins in determining atherosclerosis is established and in this context oxidised lipoproteins (particularly LDL) seem to be the main culprits. All lipoproteins contain cholesterol and its esters.
As for the rest of the lipids, cholesterol (Ch) is subjected to oxidation phenomena that derive from both enzymatic and non-enzymatic processes, which can be of endogenous origin or due to an ingestion of oxidised cholesterol that is generated during food storage and cooking processes.
In general, the oxidative processes of cholesterol lead to the formation of oxysterols (OxChs), at least four of which are endogenous in origin. Three of these, respectively 7α-hydroxycholesterol, 24-hydroxycholesterol, 27-hydroxycholesterol, derive from the oxidative process that takes place in the cytochromes P450 due to the formation of bile salts, while the fourth, 4β-hydroxycholesterol, is not correlated to the synthesis of these endogenous salts. An intermediate between Ch and 7β-hydroxycholesterol is represented by 7β-hydroperoxycholesterol, which is a classic hydroperoxide that can carry out the Fenton reaction and is thus capable of propagating OS.
A part of the OxChs can be deemed physiological and functional due to the polarity/mobility that allows them to cross the membranes more easily (with a speed 3 orders of magnitude higher than that of Ch, so as to represent a means of eliminating the cell excess of Ch. However, their excess is also harmful and such as to rapidly trigger the reactive cell self-destruction (apoptosis) or aggression system on the part of the macrophages, which will consequently result in removal of the cell itself.
It should lastly be noted that food cholesterol, which intake is on average in the order of a hundred or so milligrams/day, can already be in oxidised form, due to inadequate (and/or prolonged) storage of food, as well as following cooking of the food. In this form, as is moreover the case for all lipids, it is partially absorbed and incorporated into the chylomicrons that form inside the intestinal cells (enterocytes), and unique among oxidised lipids, can be transferred to all lipoproteins (the other oxidised lipids are limited to chylomicrons and remnants). OxChs are therefore used in the formation of variable-density lipoproteins (VLDL, HDL), which will already be partially oxidised and can trigger further oxidation processes by propagation (being circulating). It should be noted that, in terms of oxidation, it is important to consider total VLDL cholesterol, IDL (remnants), LDL or HDL, without distinction in that any cholesterol structure can be oxidised, as such or in esterified form.
Given the enormous importance of cholesterol, both in the structural and metabolic sense, it is equipped with a broad defence system.
The first line of defence is its esterification with fatty acids (of different carbon chain length). This process allows the deposition of cholesterol inside the lipoproteins. In normal conditions, Ch is collected both as such and in its esterified form by hepatic and intestinal ACAT A: Cholesteryl Acyl Transferase) and is then conveyed to the high-density lipoproteins (HDL). These will subsequently transfer it through transporter proteins (i.e. Cholesteryl Ester Protein Transporters or CEPT) to the other, more low-density lipoproteins (primarily to VLDL and to LDL), exchanging it with the triglycerides. The transfer of the triglycerides, which will be conveyed (by the same HDL) towards the liver with the esterified cholesterol residue, is operated in this way, both as such and as ChOx.
The second line of defence is represented by the antioxidant enzyme systems present in the HDL themselves, the paraoxonases (PON). These are assisted by the circulating antioxidant network (AO), which comprise a varied series of derivatives ranging from albumin, uric acid and the vitaminic (e.g. Vit E and Vit C) or non-vitamin (e.g. polyphenols) antioxidants taken with food.
The third line of defence is represented by the antioxidant systems of the cellular compartment, primarily represented by free glutathione (GSH) and by classic intracellular antioxidants (e.g. B-group vitamins, coenzyme Q10, lipoic acid), as well as by enzymatic antioxidants such as catalases, peroxidases, glutareductases.
Since ROOH are relatively stable, they represent an oxidative threat for both structural and metabolic cholesterol. However, the oxidative process must be seen as a ratio of the oxidative entities to the oxidisable entities. This process follows the stoichiometric rule for which the concentrations of both the molecular components involved in the oxidative and protective relationships must be evaluated.
The aim of the present invention is thus to identify a method that allows the risk caused by an altered oxidative balance to be reliably, reproducibly, repetitively and economically evaluated.